TRAIL is a potentially very important therapeutic as it shows selectivity for inducing apoptosis in cancer cells whilst normal cells are refractory. TRAIL binding to Death Receptors-4 and -5 leads to recruitment of caspase-8 and classical activation of downstream effectors caspases. As with many drugs, TRAIL's usefulness is limited by resistance, either innate or acquired. In Aim 1, we propose the development of a novel 1536-well high-throughput screening (HTS) strategy for identifying TRAIL-sensitizing agents that act solely in a caspase-8 dependent manner, rather than through general toxicity. This will be accomplished by utilizing a TRAIL resistant cell line lacking caspase-8 (NB7) compared to the same cells reconstituted with the protein. Importantly, false positive hits can easily be weeded out in this assay due to their activity in cells lacking caspase-8-inducible activity. Our screenig strategy's real power is based in its ability to identify sensitizing agents that act specifically through the caspase-8 apoptotic axis rather than through generalized toxicity. We further note that our system can identify agents acting on any molecule or process involved in the TRAIL-caspase-8 extrinsic death pathway and not solely on caspase-8 itself. In our preliminary studies, we have developed the ATP consumption CellTiterGlo cell survival assay in a 384-well system with high statistical rigor (Z' = 0.85 for the NB7+Caspase-8 cells). Screening of the library of pharmacological agents (LOPAC) has already been performed as a proof-of-concept to discover TRAIL sensitizers whose mechanism is caspase-8 mediated. Use of the proteasome inhibitor MG-132 as a TRAIL sensitizing positive control agent allows for monitoring of sensitivity across all platforms used. Here, we propose to develop this assay further and optimize the system for highly automated HTS, and subsequently to screen the ~330,600 MLSMR collection with a view to identifying molecular probes that may assist in the interrogation of signaling pathways relevant to human disease and to potentially identify novel therapeutic intervention points. In Aim 2, secondary analysis of any hits obtained will be carried out by the CaspaseGlo assay system, in order to measure downstream effectors caspase activity. Again this assay will be developed, optimized and miniaturized to 1536-well plate format and statistical validity will be re confirmed by calculating Z' values. Change to 1536-format will result in reductions in reagent use of 75%. All assay systems will be assessed for signal sensitivity with high Z' values. Assessment of compounds ability to sensitize TRAIL induced caspase-8 activity should provide initial clues as to say probes mechanisms of action, which can then be further assessed using standard molecular biology techniques during tertiary analysis. Biochemical analysis such as immunoprecipitation and immunoblot interrogation of the known signaling components coupled with proteomic studies, gene expression analysis and functional genomics screens are expected to provide valuable mechanism of action data. In sum, we propose a HTS of the expanded ~330,600 MLSMR collection of compounds with a unique screening strategy optimized for the detection of caspase-8 pathway specific TRAIL sensitizing agents. Such agents are expected to lack generalized cellular toxicity, and could therefore function as attractive lead compounds for further development. Additionally, such agents can be used as tool compounds to further characterize and study cellular survival and death pathways that are differentially regulated in normal and cancer cells.